Cell wall synthesis and remodeling are essential processes central to bacterial growth and division. The long-term goal of this basic-science proposal is to fill in major gaps in fundamental knowledge about the functions and regulation of the individual penicillin-binding proteins (PBPs) that synthesize peptidoglycan (PG) in bacterial cell walls. A major unmet challenge in mapping PBP activation and localization, as well as the relationships between the PBPs and their associated regulatory proteins, has been the inability to assess the functional state of the separate PBP homologs that are present in each bacterial species over the course of growth and division. We propose to generate new tools and approaches that will contribute to a comprehensive understanding of PBP function and regulation by pursuit of the following two Aims. Aim 1: Develop selective activity-based probes for each PBP homolog in a Streptococcus pneumoniae model utilizing both known and novel electrophilic scaffolds, in combination with protein crystallography and molecular modeling. Design of probes to target each PBP homolog in an organism requires the identification of scaffolds that selectively inhibit each enzyme. We have mapped the PBP selectivity of a library of ?-lactams, providing a solid foundation for the development of activity-based probes; however, many PBPs are poorly inhibited by existing ?-lactams. To address this additional challenge, we will employ molecular modeling and structural biology studies, as well as our newly identified PBP-selective ?-lactone scaffold for the development of a suite of probes for the PBPs of S. pneumoniae, which is a powerful cell biology model to establish and validate the use of this new type of probe. Aim 2. Map the localization, timing, and regulation of the transpeptidase activity of specific PBPs by using activity-based probes and complementary approaches. We will use the specific activity-based probes from Aim 1 to answer fundamental questions about the function, localization, and interactions of the PBPs in a S. pneumoniae model; these questions cannot be answered with existing strategies. In this Aim, we will use cutting-edge high-resolution microscopic techniques to determine the spatial and temporal distributions of specific, functional PBPs at different stages of the S. pneumoniae cell cycle. In addition, we will determine how cellular amounts of active PBPs are altered in mutants defective in putative PBP regulators. Finally, we will determine the interactors of PBPs by using both activity-based and complementary approaches. This grant will answer fundamental questions about PBP function and spaciotemporal regulation in a ?superbug? S. pneumoniae model and provide new tools and approaches for dissecting PG synthesis in other eubacteria, and thereby lead to a deeper understanding of this complex process in general. In addition, the new knowledge produced by this proposal will reveal novel targets and steps in PG synthesis that may be exploited as vulnerabilities for future drug development to combat the increasing emergence of antibiotic-resistant bacterial pathogens.